目的 建立替诺福韦酯（TDF）的代谢产物替诺福韦（TFV）、替芬泰（Y101）及其代谢物M8的LC-MS/MS分析方法，基于大鼠体内药动学、肾排泄及体外肾切片摄取模型研究Y101与TDF的药物-药物相互作用（DDI）。方法 ①药动学实验：SD雄性大鼠随机分为3组，分别单次ig给药Y101（60 mg·kg^-1）、TDF（30 mg·kg^-1）及TDF（30 mg·kg^-1）＋Y101（60 mg·kg^-1），通过LC-MS/MS方法测定给药后血浆中Y101、M8、TFV浓度，并采用非房室模型统计矩法计算药动学参数。②肾排泄实验： SD雄性大鼠随机分为3组，分别单次iv给药Y101（25 mg·kg^-1）、TDF（30 mg·kg^-1）及TDF（30 mg·kg^-1）＋Y101（25 mg·kg^-1）。利用LC-MS/MS方法测定尿样中Y101、M8、TFV浓度，分析药物及代谢物的累积排泄率；③肾切片实验：大鼠肾切片分别在含有M8（5.0 μmol·L^-1）、TFV（10 μmol·L^-1）、TFV（10 μmol·L^-1）＋Y101（2.0 μmol·L^-1）和TFV（10 μmol·L^-1）＋M8（5.0 μmol·L^-1）的药液中孵育一定时间后，收集样品经适当处理后利用LC-MS/MS方法测定肾脏对TFV、Y101、M8的摄取量。结果 ①药动学实验：对Y101及M8开展部分方法学验证，对TFV开展全面的方法学验证，验证结果表明LC-MS/MS方法专属性强、灵敏度高。采用LC-MS/MS方法测定大鼠血浆中的TFV、Y101和M8，发现与单独用药组相比，Y101＋TDF组大鼠血浆中TFV、Y101和M8药时曲线下面积（AUC_0-t和AUC_0-∞）显著性增加，血浆清除率（CL_P）显著性减小。②肾排泄实验：Y101＋TDF组的尿累积排泄分数低于单独用药组，且TFV、M8均出现显著性降低。③肾切片实验：TFV＋M8组与单独孵育组相比，肾切片对TFV或M8的摄取量均显著性下降。结论 TDF的代谢产物TFV与Y101的代谢产物M8可能通过竞争性抑制有机阴离子转运体（3 OAT3），对TFV、M8的血药浓度及肾排泄造成影响，提示临床合用需要进行剂量调整。

Objective To establish LC-MS/MS methods for the analysis of tenofovir (TFV), bentysrepinine (Y101) and its metabolite M8. The drug-drug interactions between Y101 and tenofovir disoproxil fumarate (TDF) and its mechanism were studied by using the model of in vivo pharmacokinetics, renal excretion and in vitro uptake in kidney slices. Methods ① Pharmacokinetic experiments: SD male rats were randomly divided into three groups: rats were orally given Y101 (60 mg·kg^-1), TDF (30 mg·kg^-1) or TDF (30 mg·kg^-1) + Y101 (60 mg·kg^-1), respectively. Plasma concentrations of Y101, M8 and TFV were determined by LC-MS/MS methods, and pharmacokinetic parameters were calculated by Phenix WinNonlin with non-compartmental analysis. ② Renal excretion experiments: SD male rats were randomly divided into three groups: Rats via tail vein were injected with Y101(25 mg·kg^-1), TDF (30 mg·kg^-1) or TDF (30 mg·kg^-1) + Y101 (25 mg·kg^-1), respectively. The concentrations of Y101, M8 and TFV in urine samples were determined by LC-MS/MS methods, and the cumulative excretion rates of drugs and their metabolites were calculated. ③ Kidney slices experiments: Rat kidney slices were incubated in buffer containing M8 (5.0 μmol·L^-1), TFV (10 μmol·L^-1), TFV (10 μmol·L^-1) + Y101 (2.0 μmol·L^-1) or TFV (10 μmol·L^-1) + M8 (5.0 μmol·L^-1) for a certain period of time, respectively. The samples were collected and properly treated, and then the renal uptakes of TFV, Y101 and M8 were determined by LC-MS/MS methods. Results ① Pharmacokinetic experiments: A full validation of TFV and a partial validation of Y101 and M8 were carried out. These results showed that the LC-MS/MS methods were highly specific and sensitive. The concentrations of TFV, Y101 and M8 in rat plasma were determined by validated LC-MS/MS methods. It was found that compared with the Y101 or TDF treatment group, the area under the curve (AUC_0-t and AUC_0-∞) of TFV, Y101 and M8 in rat plasma increased significantly and the plasma clearance rate (CL_P) decreased significantly in the group of TDF + Y101. ② Renal excretion studies: The results showed that the cumulative urinary excretion of TFV and M8 in the TDF+Y101 group was significantly lower than the Y101 or TDF alone group. ③ Kidney slices experiments: Compared with the TFV or M8 alone group, the uptakes of TFV or M8 decreased significantly in the TFV + M8 group. Conclusion TFV and M8 may have an impact on the blood concentrations and renal excretion of TFV and M8 through competitive inhibition of OAT3, suggesting that the dose needs to be adjusted in clinical combination.

R969.2

国家自然科学基金青年基金项目(82104284)